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Biochemistry 1991, 30, 2600-2612
Communication between the Active Sites in Dimeric Mercuric Ion Reductase: An Alternating Sites Hypothesis for Catalysis? Susan M. Miller,*,* Vincent Massey,t Charles H. Williams, Jr.,fs David P. Ballou,l and Christopher T. Walsh" Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 481 09, VA Medical Center, Ann Arbor, Michigan 481 05, and Department of Biological Chemistry and Molecular Pharmacology, Haruard Medical School, Boston, Massachusetts 021 I5 Received August 31, 1990; Revised Manuscript Received December 12, I990
ABSTRACT: Mercuric reductase, a flavoprotein disulfide oxidoreductase, catalyzes the two-electron reduction of Hg(I1) to Hg(0) by NADPH. As with all the members of this class of proteins, the enzyme is a dimer of identical subunits with two active sites per dimer, each composed of one FAD and catalytically essential residues from both subunits. In the enzyme from Tn501, these residues include, a t a minimum, FAD and cysteines 135 and 140 from one subunit and cysteines 558' and 559' from the other. With this sort of active site arrangement, the enzyme seems perfectly set up for some type of subunit communication. In this report, we present results from several titrations, as well as kinetics studies, that, taken together, are consistent with the occurrence of subunit communication. In particular, the results indicate that pyridine nucleotide complexed dimers of the enzyme are asymmetric. Since the EH,-NADPH complex of the enzyme is the relevant reductant of Hg(II), these observations suggest that the enzyme may function asymmetrically during catalysis. An alternating sites model is proposed for the catalytic reduction of Hg(II), where both subunits of the dimer function in catalysis, but the steps are staggered and the subunits reverse roles after part of the reaction. An attractive feature of this proposal is that it provides a reasonable solution to the thermodynamic dilemma the enzyme faces in needing to both bind Ilg(I1) very tightly and reduce it.
M e r c u r i c reductase is a flavoprotein that catalyzes the conserved in all sequences of mercuric reductases reported to reduction of Hg(SR), by NADPH as the final step in the date (Brown et al., 1983; Misra et al., 1985; Griffin et al., detoxification of organomercurials and mercuric ion in a wide 1987; Wang et al., 1989) and which is absent from the other members of this class of flavoproteins, confers on mercuric variety of bacterial strains (Foster, 1983; Summers, 1986; Silver & Misra, 1988). In its primary sequence and in its reductase the unique ability to reduce the toxic Hg(I1) ion rather than merely bind it. spectral properties mercuric reductase is strikingly similar to the pyridine nucleotide disulfide oxidoreductases, including Along with its homologous relatives, mercuric reductase is glutathione reductase, lipoamide dehydrogenase, and trypaa dimer of identical subunits with one active site (FAD) per nothione reductase (Fox & Walsh, 1982; Williams, 1991; subunit. Crystal structure determinations of glutathione reductase (Thieme et al., 1981; Karplus & Schulz, 1987) and Shames et al., 1986). These enzymes all contain both FAD and a redox-active disulfide at the active site (Williams, 1991), lipoamide dehydrogenase (Schierbeek et al., 1989) have shown which is composed of Cys,,,Cys,, in Tn501-encoded mercuric that each active site in the dimeric enzymes is composed of reductase (Fox & Walsh, 1983; Brown et al., 1983). Unlike residues from both subunits: FAD and redox-active disulfide from one subunit, and an essential histidine acid/base catalyst the other cnzymes, however, mercuric reductase contains two from the carboxy-terminal region of the other subunit. On additional thiol pairs, one in an 80-residue N-terminal exthe basis of this structure and the extensive similarities between tension (CysloCys13) and one near the C-terminus ( C y ~ , & y s ~in~Tn501-encoded ~ mercuric reductase and glutathione reductase, Brown et al. enzyme) (Brown et al., 1983). (1983) speculated that the C-terminal cysteines of one moThe role of these three cysteine pairs has been examined by nomer of mercuric reductase may be involved in Hg(I1) physical and chemical techniques with the wild-type enzyme binding at the FAD/dithiol site of the other monomer of the and with a variety of site-directed mutants of the enzyme dimer. Distefano et al. (1990) have recently substantiated this having one or more of these six cysteines changed to alanine hypothesis for the enzyme from Tn501 by generating active or serine. These studies showed that the amino-proximal thiol heterodimers from two inactive site-directed mutants, one pair (CyslOCysl3)is not essential for efficient reduction of lacking the active-site dithiol (Ala135Alal,o),and the other Hg(l1) ( F o x & Walsh, 1983; Moore & Walsh, 1989). lacking the carboxy-proximal dithiol (Ala5,8Ala5,9). PrelimHowever, the carboxy-proximal thiol pair, Cy~,,,Cys,,~,clearly communicates with the active-site dithiol pair, C y ~ , ~ ~ C y s , ~inary ~ , crystal structure data have further verified this hypothesis for mercuric reductase isolated from Bacillus sp strain RC607 and plays an essential role in binding and positioning Hg(I1) (Schiering et al., 1990). for reduction (Miller, et al., 1989; Moore & Walsh, 1989). The requirement for this monomer-monomer interaction This carboxy-proximal cysteine pair, which is completely in normal activity raises the question of whether cooperative or half-sites behavior is present and, if so, whether it is im* To whom correspondence should be addressed. 'This work was supported by National Institutes of Health Grants portant in the normal functioning of the enzyme. No clear GM I I106 (V.M.), GM 21444 (C.H.W.), GM 20877 (D.P.B.), and GM evidence for such behavior has been reported for glutathione 21643 (C.T.W.). reductase. In lipoamide dehydrogenase, only two examples *University of Michigan Medical School. of nonidentical behavior have been reported. For wild-type SVA Medical Center. enzyme, van Muiswinkel-Voetberg and Veeger (1 973) reported '1 Harvard Mcdical School. 0006-296019 1 10430-2600$02.50/0
0 1991 American Chemical Societv
Biochemistry, Vol. 30, No. 10, 1991
Half-Site Behavior in Mercuric Reductase
2601
Scheme I: Redox States of Wild-Type Mercuric Reductase"
S(H) HS SH Nonactivated Eo x
Activated
HS
EHZ
SH
HS
EH4
Eo,
'The dashed-line symbol between FAD and S- represents the charge-transfer interaction.
that binding of NAD+ to the oxidized form of the enzyme occurs in a nonuniform manner, indicating the presence of two different NAD+ binding sites. With lipoamide dehydrogenase that is monoalkylated on the interchange thiol, binding of NAD+ gives rise to spectral changes indicative of the formation of a flavin C(4a)-thiol adduct; however, the changes indicate only 50% of the adduct is formed (Thorpe & Williams, 1981). With mercuric reductase, two early observations with the wild-type enzyme suggested some type of site-site interaction: ( I ) Purified enzyme was found to bind ca. 0.7 equiv of N A D P per dimer after exhaustive dialysis against NADP+-free buffer, suggesting the presence of one tighter and one weaker binding site per dimer (Fox & Walsh, 1982). (2) Reaction of the two-electron-reduced enzyme EH2 with [14C]iodoacetamide resulted in a biphasic loss of Hg(I1) reductase activity concomitant with incorporation of radioactivity. However, spectral changes and the accompanying increase in transhydrogenation activity appeared to be complete in the fast phase of inactivation (Fox & Walsh, 1983). In the course of our studies on the properties of wild-type mercuric reductase and various Cys to Ala and Cys to Ser site-directed mutants, we have observed a variety of phenomena indicating nonequivalence in the properties of the subunits of the dimer. We present here examples of simple binding reactions, redox reactions, and kinetics studies of wild-type and mutant forms of the enzyme, all of which clearly exhibit differential behavior of the subunits. Based on these results, an alternating sites model for catalytic reduction of Hg(l1) is developed, and the advantages of this type of model are discussed. EXPERIMENTAL PROCEDURES Materials. Wild-type mercuric reductase was purified from Escherichia coli W31 IO lacP containing the plasmid pPSOl [which contains the Tn501 mercuric reductase (merA) gene] as previously described (Miller et al., 1989). Specific Cys to Ala and Cys to Ser mutants of mercuric reductase were purified from the above strain of E. coli containing the modified plasmids as previously described (Moore & Walsh, 1989; Distefano et al., 1989; Miller et al., 1990; Schultz et al., 1985). Methods. All experiments were conducted in 50 mM potassium phosphate buffer, pH 7.3, containing 0.3 mM EDTA (standard buffer), unless otherwise noted. UV-vis spectra were recorded with Cary I7 or 219 double-beam spectrophotometers or a Hewlett-Packard 8452A diode array spectrophotometer. Fluorescence spectra were recorded with a scanning ratio spectrofluorometer built by Gordon Ford and Dr. David Ballou at the University of Michigan. Rapid reaction studies were carried out with two different stopped-flow spectrophotometers with UV-vis absorbance capabilities: (1) One is equipped with a monochromator drive either for single-wavelength studies or for scanning spectra over a 7-s time scale (2-cm optical path) as previously described (Beaty & Ballou, 1981). (2) The other is equipped with a Tracor-Northern diode array detector for obtaining spectra of reaction intermediates on a
millisecond time scale. Rapid reactions studied by fluorescence were carried out with a stopped-flow spectrofluorometer as previously described (Brissette et al., 1989). Anaerobic titrations were conducted in cuvettes with two side arms similar to those previously described (Williams et al., 1979). Samples were made anaerobic by subjecting the sample to several vacuum/nitrogen (or argon) equilibration cycles. Gases were made anaerobic by passing over a heated column of Ridox (Fisher). Samples of wild-type EH2 for titration with NADPH and NADP+ were prepared by anaerobic reduction of E,, with sodium dithionite. Titrations were made by using Hamilton gastight syringes equipped with threaded plungers. Redox Titrations. Redox titrations were performed by using a new method (Massey, 1990). Xanthine oxidase (90 nM) and xanthine (670 p M ) (go = -366 mV; Green, 1934; Clark, 1960) were used as a source of electrons with a redox dye acting both as an electron mediator between the proteins and as a redox indicator. Methyl viologen (go = -449 mV) was used for the Ser,,, mutant mercuric reductase, and diquat (go = -361 mV) was used for the Ser,,, mutant. Experimentally, the enzyme of interest, an appropriate buffer, xanthine, and the indicator dye are placed in an anaerobic cuvette, with xanthine oxidase in a side arm. After anaerobiosis, xanthine oxidase is tipped in to start the reduction. The absorbance at both the A, of the enzyme and an appropriate wavelength for determining the amount of reduced indicator were then monitored. Since the xanthine/xanthine oxidase method described here involves the continuous generation and equilibration of reducing equivalents, there is the possibility that incomplete equilibration would occur in the system whose potential is being measured. In order to avoid such complications, the concentration of xanthine oxidase added was adjusted such that reduction of the enzyme under study occurred over a period of 4-6 h, and halving the amount of xanthine oxidase added did not change the log/log plots used to determine redox potential. The enzyme potentials were calculated as derived in the Appendix. RESULTS Since differential behavior has been observed in a number of different forms of both wild-type and mutant mercuric reductases, a description of the redox states of the enzyme, as well as the nomenclature of the mutant species, is presented here. Scheme I shows the several redox states of wild-type enzyme. Oxidized enzyme (E,,) contains FAD and a redoxactive disulfide ( C y ~ ~ ~ ~ C as y swell , ~ )as, an auxiliary cysteine pair, CysS5, and CysSs9,(primes indicate residues from the other subunit of the dimer). In enzyme fully activated toward Hg(I1) reduction, the auxiliary pair exists as a dithiol, whereas in nonactivated enzyme (Sandstrom & Lindskog, 1987), it is a disulfide (Miller et al., 1989). E,, forms of the enzyme do
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Miller et al.
Biochemistry, Vol. 30, No. 10, 1991
not participate in turnover (Miller et al., 1986); rather, both disulfides must be fully reduced in a priming step to form active EH2. In this form the Cys,,, thiolate is highly stabilized (pK, N 5.0; unpublished results) and participates in a charge-transfer interaction with FAD, giving rise to the characteristic absorption spectrum of this species (e.g., Figure 2). Further addition of two electrons leads to formation of EH4 with reduced flavin and all four thiols reduced. Mutant mercuric reductases used in the present studies include those with mutations of either the N-terminal cysteines, the activc-site disulfide, the C-terminal cysteines, or a combination of the active-site and C-terminal pairs. The di(A1a to Cys) mutant, Ala,oAla,,, is a wild-type mimic, as was expected from previous proteolysis studies (Fox & Walsh, 1983). For mutations of the enzyme in the active-site and/or the C-terminal cysteines, we use a nomenclature where the one-letter codes for the cysteines or their replacements are listed in the order in which they come in the amino acid sequence (135, 140, 558, 559) (Distefano et al., 1990). Thus, wild-type enzyme (or the AlaloAla,3)is referred to as CCCC enzyme. A mutant having only Cys,,, changed to alanine is referred to as ACCC enzyme, etc. Mutants having either or both cysteines 135 and 140 replaced by serine or alanine retain only the flavin as a redox center; they can only exist in two redox states, E-FAD and E-FADH(,,. These mutants can be grouped into two types: those retaining Cys,,, which are EH2 mimics since they still have the thiolate/flavin charge-transfer interaction that is present in wild-type EH,, and those that have either serine or alanine at residue 140, which are E,, mimics because they lack the charge-transfer interaction. Evidence f o r Differential Behavior in Ligand Binding Processes Spectral Changes upon Titration with N A D P of the E,, Forms of Wild-Type and Several Mutant Mercuric Reductases. Mutations of the various cysteines in mercuric reductase generate minimal effects on the ability of the enzymes to bind pyridine nucleotides (Schultz et al., 1985; Distefano et al., 1989; Moore & Walsh, 1989). Hence, the E,, form of all mercuric reductases binds N A D P to form an enzymeproduct complex. The stoichiometry of binding has been determined to be 1 equiv of NADP+ per FAD for the AlaloAla,, enzyme at pH 7.3. Thus, when 50.2 pM NADP+ was added to 17.2 pM enzyme monomer (final concentrations), and the enzyme was concentrated over a Centricon-30 membrane, 34 pM NADP' was found in the filtrate, giving, by difference, 16.2 pM bound NADP' or 0.94 equiv per FAD. In spite of thc simple stoichiometry of binding, the accompanying spectral changes are unusual. As illustrated in Figure IA for the Ala,oAla13enzyme at pH 7.3, binding of NADP+ causes a dramatic change from the resolved UV-visible absorption spectrum of E,, to one with little or no resolution in the 400-550-nmregion. In contrast, the fluorescence excitation spectrum, Figure 1 B, merely decreases in intensity, to a final value of 41-42% of the initial value, while maintaining the initial resolved form of pure E,,. The same behavior has also been observed for this mutant at pH 5.1, and for wild-type enzyme and the CCCA, CCAC, CCAA (Moore, 1989), AACC, and CACC (Distefano 1989) mutant enzymes at pH 7.3. In each case the final absorbance spectrum loses resolution while the final fluorescence excitation spectrum does not change in shape but is reduced in intensity to between 40 and 45% of the starting value. Fluorescence excitation spectra generally mimic the absorption spectrum of the fluorescent species. Hence, the above results are quite unexpected and suggest that NADP+-bound
0.10 Q)
0
S
W
d
I
0 0.05 v)
d
a 0.00 100
1
"
'
n
I
~
"
l
"
'
-.
0
"
300
"
"
" ~ 400
"
" " 500
'
~ 600
700
Wavelength (nm) 1 : Binding of NADP+ to the E, form of the Alal&Ial3mutant mercuric reductase at pH 7.3. The AlaloAla,3enzyme (10.6 p M ) in standard buffer was titrated with NADP' and both the UV-visible FIGURE
absorbance spectra and the fluorescence excitation spectra (emission A-, = 520 nm) were recorded at 4 O C . The sample was filtered before titration and after each addition through the same Gelman Acrodisc LC13 filter (0.45 pm). Panel A: Absorbance spectra shown are (upper solid) starting enzyme, (- - -) end point in the presence of 107.1 pM NADP+, (lower solid) 0% starting enzyme, and (-) end point minus 0.5X starting enzyme. Upper inset shows the fractional change in absorbance at 456 nm (0)and 516 nm (A)vs equivalents of NADP+ added per FAD as well as a line of unit slope. Lower inset shows E,, form of the CSCC enzyme (8.3 p M ) in the presence of ( - - - ) 0 N A D P and (-) 90.2 ~ L M NADP+. Panel B Fluorescence excitation spectra (Aem 525 nm) of (-) starting enzyme, (- - -) end point in the end point normalized to the presence of 107.1 pM NADP', and initial intensity at 456 nm. Inset shows the fractional change in fluorescence intensity with excitation at 456 nm (0) vs the equivalents of NADP' added per FAD as well as a line of unit slope. (a-)
enzyme is a composite of two species, one with a dramatically altered UV-visible spectrum and totally quenched FAD fluorescence and one with essentially no change in the UVvisible spectrum and only a slight quenching of the fluorescence. Assuming that the final fluorescence excitation spectrum is the same as that of the UV-visible absorbance spectrum for one site of the enzyme, we can estimate the UVvisible spectrum of the altered site by subtraction from the final unresolved spectrum of the NADP+-saturated species. Figure 1A also shows the spectrum of 50% of the unliganded enzyme and the result of subtracting this from the spectrum of E,,-NADP+. The difference spectrum (dotted line) suggests that the altered site has a spectrum similar in shape to uncomplexed enzyme but shifted ca. 15 nm to longer wavelength. Corroboration of the above analysis comes from the binding of NADP' to the one anomalous mutant in this reaction, the CSCC enzyme. In this case, complexed enzyme exhibits a spectrum nearly identical in shape with that of the unliganded enzyme but shifted to longer wavelength by 16 nm and lowered in extinction (lower inset, Figure IA). The nature of these
i/
Biochemistry, Vol. 30, No. 10, 1991
Half-Site Behavior in Mercuric Reductase 0.1 60
2603
0.085
a0.120
a 0 c
0
c U
0.1 5
0.045
;Oslo
0.080 0
0
